WO2002070715A1 - Lysozime de type g humain, sa sequence codante, son procede de preparation et ses utilisations - Google Patents

Lysozime de type g humain, sa sequence codante, son procede de preparation et ses utilisations Download PDF

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Publication number
WO2002070715A1
WO2002070715A1 PCT/CN2001/001176 CN0101176W WO02070715A1 WO 2002070715 A1 WO2002070715 A1 WO 2002070715A1 CN 0101176 W CN0101176 W CN 0101176W WO 02070715 A1 WO02070715 A1 WO 02070715A1
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Prior art keywords
lyg2
sequence
polynucleotide
polypeptide
expression
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PCT/CN2001/001176
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English (en)
Chinese (zh)
Inventor
Long Yu
Yong Zhao
Peirong Hu
Lisha Tang
Shouyuan Zhao
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Long Yu
Yong Zhao
Peirong Hu
Lisha Tang
Shouyuan Zhao
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Application filed by Long Yu, Yong Zhao, Peirong Hu, Lisha Tang, Shouyuan Zhao filed Critical Long Yu
Priority to US10/469,602 priority Critical patent/US6943244B2/en
Publication of WO2002070715A1 publication Critical patent/WO2002070715A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases

Definitions

  • the present invention provides a novel human goose-type lysozyme nucleic acid and amino acid sequence, and provides a method for diagnosing, treating, and preventing autoimmune system / inflammation, kidney, adrenal diseases and tumors. Background technique
  • Lysozyme is a family of enzymes that can catalyze the hydrolysis of certain mucopolysaccharides of the bacterial cell wall, especially the ⁇ -1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine, and can cause bacterial lysis. Lysozyme is found in a variety of organisms such as viruses, birds and mammals. In humans, lysozyme is found in the spleen, lungs, kidneys, white blood cells, plasma, saliva, milk, tears and cartilage. (Online Mendelian Inheritance in M ⁇ (OMIM) 153450 Lysozyme; Weaver, L. H. et al. (1985) J. Mol. Biol. 184: 739-741).
  • Type C and type G lysozyme have similar three-dimensional structures but different amino acid sequences. (Nakano, T. and Graf, T. (1991) Biochim. Biophys. Acta 1090: 273-276). In chicken, both types of lysozyme are present in neutrophils, while only C is present in egg white. Type lysozyme.
  • Homologous G-type lysozyme was found in chicken, black swan, goose and ostrich.
  • the conserved residues include three catalytic center residues, Glu99, Aspl2, and As P 123 (counted by chicken G-type lysozyme precursors) and 4 cysteine residues. These 4 cysteine residues are in the black swan G-type Two disulfide bonds are formed in lysozyme.
  • isoenzymes have been found in rabbits, including leukocyte type, digestive tract type, and perhaps lymphoepithelial cell type (OMIM153450, supra; Nakono (1991) supra; and GenBank g 1310929).
  • Lysozyme is associated with many diseases. Nakano (supra) believes that lysozyme may play a role in the host defense system. Older rabbits with hereditary lysozyme deficiency have increased sensitivity to infections, especially the high sensitivity of subcutaneous abscesses (0MIM153450, supra). Mutations in the human lysozyme gene cause hereditary systemic amyloidosis. The disease is a rare dominant autosomal disease with amyloid deposits in the viscera, including the kidneys, adrenals, spleen, and liver, and is usually fatal before the age of 50. The amyloid deposits contain lysozymes in which amino acids are replaced.
  • Renal amyloidosis is the most common and the most severe organ involvement (Pepys, MB et al. (1993) Nature 362: 553-557; OMIM 105200 Fami lial Visceral Amyloisosis; Cotran, RS et al. (1994) Robbins Pathologic Basis of Disease, WB Saunders Company, Philadelphia, Pa., Pp. 231-238)
  • G-type lysozyme is detected in promyelocytic cells of birds with myeloid leukemia virus carrying the L106 mutant of the v-myb oncogene. Expression (Nakano, T. and Graf. T. (1992) Oncogene 7: 527-534; and Nakano (1991) supra.).
  • the present invention provides a new human G-type lysozyme and its encoding polynucleotide, which provides a new approach for the diagnosis, treatment and prevention of autoimmune / inflammatory, kidney, adrenal-related diseases and cancer.
  • the present invention also provides a fully purified variant protein having at least 90% identity with the sequence of the amino acid sequence or fragment thereof in SEQ ID No. l.
  • the invention also provides an isolated and purified polynucleotide, the 10 polynucleotide encodes a polypeptide containing the amino acid sequence in SEQ ID No. 1 or a fragment sequence thereof.
  • the present invention also provides an isolated and purified polynucleotide variant that is at least 70% identical to a polynucleotide encoding a sequence containing the amino acid sequence in SEQ ID No. 1 or a fragment thereof.
  • the invention also provides an isolated and purified polynucleotide capable of interacting with a polynucleoside encoding a polypeptide containing the amino acid sequence in SEQ ID No. 1 or a fragment sequence thereof under stringent conditions.
  • Acid hybridization also provides a polynucleotide complementary to a polynucleotide encoding a polypeptide containing the amino acid sequence in SEQ ID No. 1 or a fragment sequence thereof.
  • the present invention also provides an isolated and purified polynucleotide, which contains the polynucleotide sequence 1-846 or 114-662 in SEQ ID No. 2 or a fragment sequence thereof. .
  • the present invention also provides an isolated and purified polynucleotide variant having at least 70% identity with a 10-polynucleotide containing the polynucleotide sequence or fragment sequence in SEQ ID No. 2 .
  • Also provided is an isolated and purified polynucleotide complementary to a polynucleotide comprising the sequence of SEQ ID No. 2 or a fragment sequence thereof.
  • the present invention also provides an expression vector containing at least one polynucleotide encoding an amino acid sequence or a fragment thereof contained in SEQ ID No. 1. On the other hand, this expression vector is contained in a host cell.
  • the invention also provides a method for preparing a polypeptide containing the amino acid sequence in SEQ ID No. 1 or a fragment thereof.
  • the steps are: (a) culturing a host cell in an environment suitable for expression of a polypeptide containing the amino acid sequence or a fragment thereof in SEQ IP No. 1, the host cell contains an expression vector, the expression vector carrying at least Polynucleotide of the amino acid sequence in ID No. 1 or a fragment thereof; (b) extracting the polypeptide from the host culture.
  • the invention also provides a pharmaceutical component, which contains a fully purified polypeptide, the polypeptide contains the amino acid sequence or a fragment thereof in SEQ ID! 0 No. l, and is connected to a suitable pharmaceutical carrier.
  • the present invention also provides a purified antibody that binds to a polypeptide comprising the amino acid sequence in SEQ ID No. 1 or a fragment thereof, and an agonist and a purified antagonist of the purified antibody.
  • the present invention also provides a method for treating or preventing an autoimmune / inflammatory disease, which method comprises: administering an effective dose of a pharmaceutical ingredient to a patient in need of treatment, the ingredient comprising the amino acid sequence of SEQ ID No. 1 or '5 fragment of purified polypeptide.
  • the present invention also provides a method for treating or preventing kidney disease, which method comprises: administering to a patient in need of treatment an effective dose of a pharmaceutical ingredient, the ingredient comprising a purification having the amino acid sequence of SEQ ID No. 1 or a fragment thereof Peptide.
  • the invention also provides a method for treating or preventing adrenal diseases, the method comprising: The patient takes an effective amount of a pharmaceutical ingredient which contains a purified polypeptide carrying the amino acid sequence of SEQ ID No. 1 or a fragment thereof.
  • the present invention also provides a method for treating or preventing cancer, the method comprising: administering an effective amount of an antagonist to a patient in need thereof, wherein the antagonist can antagonize a polypeptide having the amino acid sequence of SEQ ID No. 1 or a fragment thereof.
  • the invention also provides a method for detecting a polynucleotide encoding an amino acid in SEQ ID No. 1 or a fragment thereof in a biological sample containing a nucleic acid. Its steps are-
  • the method also includes amplifying the polynucleotide before hybridization.
  • LYG2 refers to the fully purified amino acid sequence of LYG2 or its variant form, and its source can be any species, especially mammals, including cattle, sheep, pigs, mice, horses, preferably human-derived Or any method, which can be of natural, synthetic, semi-synthetic or recombinant origin.
  • agonist refers to a molecule that, when combined with LYG2, enhances the effect of LYG2 or prolongs the effective time of LYG2.
  • Agonists can include proteins, nucleic acids, carbohydrates, or any other molecule that binds to and regulates LYG2.
  • allelic variant is another form of the LYG2 gene. Allelic variants may be caused by at least one mutation in the nucleic acid sequence, which may cause changes in the mRNA or polypeptide, which changes may or may not cause changes in the structure or function of the protein. Any particular natural or recombinant gene may be absent, one or many allelic forms.
  • “Variable" nucleic acid sequences encoding LYG2 include nucleic acid sequences containing different nucleotide deletions, insertions, or substitutions. These nucleic acids can produce a polynucleotide identical to LYG2 or a polypeptide with at least one functional characteristic of LYG2. This definition also includes some inappropriate or undesired hybridizations with allelic variants whose loci differ from the normal chromosomal loci of the polynucleotide encoding LYG2. The encoded protein can also undergo “mutation", which may contain deletions, insertions and substitutions of amino acid residues, and can also cause silent mutations, resulting in a protein that is functionally equivalent to LYG2.
  • the amino acid of LYG2 can be artificially replaced based on the polarity, charge, solubility, hydrophobicity, hydrophilicity and amphipathic properties of each residue.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • similarly hydrophobic uncharged polar amino acids including leucine, isoleucine And valine, glycine and alanine, asparagine and glutamine, serine and threonine, and phenylalanine and tyrosine.
  • amino acid refers to an oligopeptide, peptide, polypeptide, or protein sequence and includes natural and synthetic molecules.
  • fragment refers to a LYG2 fragment that is preferably at least 10 amino acids in length, or most preferably at least 14 amino acids. These fragments have some biological or immune activity of LYG2.
  • amino acid sequence refers to the amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and similar terms should not be limited to the full-length amino acid sequence of a protein molecule described herein.
  • amplification refers to the preparation of multiple copies of a nucleic acid sequence. Amplification is usually performed using PCR techniques well known in the art.
  • Antagonist refers to a molecule that reduces the effect or time of LYG2 biological or immune activity when combined with LYG2.
  • Antagonists can include proteins, nucleic acids, carbohydrates, antibodies or any other molecule that reduces the effect of LYG2.
  • antibody refers to complete molecules and fragments, such as Fab, F (ab ') 2, and Fv fragments, that can bind epitopes.
  • An antibody that binds to a LYG2 polypeptide can be obtained as a whole antigen or a small peptide-containing fragment as an immune antigen.
  • Polypeptides or oligopeptides used to immunize animals can be obtained by RNA translation or chemical synthesis, and can be linked to a carrier protein when needed. Commonly used carriers chemically coupled to peptide chains include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. The conjugated protein is ready for immunization of animals.
  • antigenic determinant refers to a segment of a molecule (such as an epitope) that is in contact with a particular antibody.
  • an epitope can compete with an antigen (that is, an immunogen that triggers an immune response) to bind antibodies.
  • antisense refers to any component that contains a nucleotide sequence that is complementary to a "sense" strand in a particular nucleotide sequence.
  • Antisense molecules can be prepared by any method including synthesis or transcription. When transferred into a cell, this complementary nucleotide can form a duplex structure with the natural sequence produced by the cell, hindering transcription or translation.
  • the "negative” strand may refer to the antisense strand, and the "positive” strand may refer to the sense strand.
  • biologically active refers to a protein that has the structure of a natural molecule, regulating or biochemical functions.
  • immunologically active refers to natural, recombinant, or synthetic LYG2 or oligopeptides associated with it, which are capable of eliciting a specific immune response and binding to a specific antibody in the appropriate animal or cell.
  • complementarity refers to the natural binding of polynucleotides formed by base-pairing. For example: 5 ⁇ -6- 3 'is combined with the complementary sequence 3'1 1 -(> 4 5'.
  • the "complementarity" between two single-stranded molecules can be “partial”, that is, only a part of the nucleic acid can be Binding can also be “complete”, meaning that the sequences of two single-stranded molecules are completely complementary.
  • a "consensus sequence” refers to a nucleic acid sequence.
  • the sequence can be a sequence obtained by repeating the sequencing to identify an uncertain base; or a nucleic acid sequence composed of multiple overlapping fragments. Some sequences are both extended and assembled to produce a consistent sequence.
  • deletion refers to a change in which one or more amino acid residues or nucleotides are missing in an amino acid sequence or nucleotide sequence.
  • derivative refers to a polypeptide sequence or polynucleotide sequence that is chemically modified.
  • a chemically modified form of a polynucleotide sequence includes amidation, acyl or amino group substitution, and the like.
  • a derived polynucleotide can encode a polypeptide that retains at least one biological or immune function of the natural molecule.
  • a derived polypeptide is a polypeptide that retains at least one biological or immune function of the original polypeptide after glycosylation or any similar process.
  • similarity refers to the degree of complementarity, which may be partially similar or completely similar. "Identity” can replace “similarity”.
  • a "fully similar” sequence refers to a partially complementary sequence that can at least partially inhibit the hybridization of a same sequence to a target nucleic acid. Inhibition of hybridization of perfectly complementary sequences to target sequences can be examined by performing hybridization analysis (Southern or Northern hybridization, etc.) under conditions of low stringency. A sufficiently similar sequence or hybridization probe can compete and inhibit the hybridization of a completely similar (identical) sequence to a target sequence under conditions of reduced stringency.
  • non-specific binding is allowed to occur under conditions of reduced stringency, Rather, under these conditions the combination of the two sequences is required to be specific (selective). Detection of non-specific binding can be achieved with another non-complementary (ie, less than 30% similarity or identity) target sequence. If there is no non-specific binding, then a sufficiently similar sequence or probe will not bind to a second non-complementary target sequence.
  • Percent identity refers to the percentage of sequence identity comparing two or more amino acid sequences or nucleic acid sequences.
  • the percent identity can be determined by a computer, such as using the PCGENE program.
  • the PCGENE program can form alignments between two or more sequences according to different methods, such as the Clustal method.
  • the Clustal algorithm divides sequences into clusters by comparing pairs of sequences. The pairs are aligned in clusters first, and then the clusters are aligned. For example, the same percentage of two amino acid sequences A and B is calculated by subtracting the sequence length of A from the number of unmatched residues in A minus the number of unmatched residues in B and dividing by all the matches in A and B , And multiply by 100.
  • Gap sequences with no or low similarity between A and B are not included in the calculation of the same percentage.
  • the percent identity between nucleic acid sequences can also be calculated using other methods in the art, such as the Jotun Hein method (see, eg, Hein, J. (1990) Methods Enzymol. 183: 626-645.). Sequence similarity can also be calculated using other methods in the art, such as changing hybridization conditions.
  • HACs Human artificial chromosomes
  • Humanized antibody refers to an animal antibody in which the amino acid sequence of the non-antigen-binding portion has been changed to be more similar to a human antibody, while still retaining the original binding function.
  • Hybridization refers to the process by which any nucleic acid strand binds to a complementary strand through base pairing.
  • hybridization complex refers to a complex produced by the formation of hydrogen bonds between two nucleic acid sequences through complementary bases.
  • Hybridization complexes can be formed in solution (eg, C. or R.t analysis) or a nucleic acid sequence in solution and an immobilized solid support (eg, paper, membrane, filter, chip, tip or glass slide, or Between any other suitable substrate on which cells or nucleic acids are immobilized).
  • insertion or “addition” refers to a change to a naturally occurring molecule that adds one or more amino acids or nucleotides to the original amino acid sequence or nucleotide sequence.
  • immune response refers to a condition related to inflammation, trauma, immune disease, infection, or genetic disease. These conditions are affected by various factors, such as the expression of cytokines, chemokines and other signaling molecules, which can affect the defense system of cells and systems.
  • micromatrix refers to the arrangement of different polynucleotides on a substrate such as paper, nylon, or any other membrane, filter, chip, glass slide, or any other suitable solid support.
  • element or “micromatrix element” refers to hybridizable polynucleotides arranged on the surface of a substrate.
  • modulation refers to a change in LYG2 activity. For example, changes in protein activity or binding characteristics, or any other increase or decrease in biological function or immune properties.
  • nucleic acid refers to nucleotides, oligonucleotides, polynucleotides, and fragments thereof. It can refer to DNA or RNA of genomic origin or synthesis. It can be single-stranded or double-stranded. It can be the sense or antisense strand. It can be a peptide nucleic acid (PNA) or any DNA-like or RNA-like substance. .
  • fragment refers to a nucleic acid sequence that can produce a polypeptide after translation. The polypeptide should retain certain functional characteristics such as antigenicity or certain domain characteristics of the original full-length polypeptide, such as the ATP binding site.
  • oligonucleotide refers to a nucleic acid sequence of at least about 6-60 nucleotides, preferably about 15-30 nucleotides, and most preferably about 20-25 nucleotides. These sequences can be used Used in PCR amplification for hybridization analysis or microarray.
  • peptide nucleotide is generally equivalent to the terms “amplification primer”, “primer”, “oligomer”, and “probe”, which are generally defined in the art.
  • PNA protein nucleic acid
  • An antisense molecule or anti-gene agent contains an oligonucleotide consisting of at least about 5 nucleotides linked to a peptide backbone consisting of amino acid residues ending with lysine. The terminal lysine makes this component soluble, and the peptide nucleic acid preferentially binds to single-stranded DNA or RNA and terminates transcriptional extension.
  • sample is used here in its broadest meaning, a biological sample that may contain a nucleotide encoding LYG2, or a fragment thereof, or LYG2 itself (which may include bodily fluids, extracts of cell chromosomes, organelles, or isolated from Cell membrane); cells, genomic DNA, RNA or CDNA, tissues, tissue blots, etc. that are present in solution or bound to a solid support.
  • binding refers to the interaction between a protein or peptide and an agonist, antibody, or antagonist, which interaction depends on the existence of a specific structure of the protein, such as an epitope or an antigenic table Bit. For example, if an antibody is specific for epitope "A”, then the presence of a polypeptide containing epitope A, or the presence of free unlabeled A, will The reaction reduces the amount of labeled A that binds the antibody.
  • stringent conditions refers to conditions under which a polynucleotide can hybridize with a claimed polynucleotide.
  • Stringent conditions can be defined by salt concentration, organic solvent concentration (e.g., formamide, temperature, or other conditions well known in the art), especially by increasing salt concentration, increasing formamide concentration, or increasing hybridization temperature to increase Stringency) concentration.
  • organic solvent concentration e.g., formamide, temperature, or other conditions well known in the art
  • substantially pure refers to a nucleic acid or amino acid sequence taken from the natural environment and isolated, and at least
  • substitution refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively.
  • transformation refers to the process by which foreign DNA enters and changes a recipient cell. According to various methods known in the art, transformation can occur under natural or artificial conditions, and foreign can be made according to any known method.
  • the insertion of a nucleic acid sequence into a prokaryotic or eukaryotic host cell depends on the type of host cell to be transformed. It may include, but is not limited to, viral infection, electroporation, electroshock, lipofection, and particle bombardment.
  • transformed cell includes cells that have been stably transformed, in which the inserted DNA can be replicated in the form of an autonomously replicating plasmid or a portion of the host chromosome, and also includes cells that are temporarily transformed and express the inserted DNA or RNA over a period cell.
  • a “variant form” of a LYG2 polypeptide refers to a chloro acid sequence that changes one or more amino acid residues. This "variant” may have “conservative” changes, which are characterized by the structural amino acid or chemical nature of the substituted amino acid (similar to that of the substituted). (Eg, replace leucine with isoleucine). More rarely, a variant may have a "non-conservative" change (eg, replacing glycine with tryptophan). Similar minor variations may include amino acid deletions or insertions, or both.
  • variant form when used in a polynucleotide sequence includes a polynucleotide sequence related to LYG2. This concept also includes “equivalent” (as defined above), “splicing", “interspecies” or “polymorphic” variants.
  • a splice variant may have significant identity with a reference molecule, but due to alternative splicing of exons during mRNA processing, it typically has a greater or lesser number of polynucleotides. Its corresponding polypeptide may have additional functional domains or domain deletions.
  • Interspecific variations are those polynucleotide sequences that differ from species to species. Because And the polypeptides they encode usually have significant amino acid identity with each other.
  • a polymorphic variation is a variation in the polynucleotide sequence of a particular gene between the same individual.
  • Polymorphic variants may also include "single nucleotide polymorphisms" (SNPs, in which the polynucleotide sequence changes by only a single base).
  • SNPs single nucleotide polymorphisms
  • the presence of SNPS may indicate, for example, a particular population, a disease state, or The tendency of a disease state.
  • a host cell refers to many such host cells
  • an antibody refers to one or more antibodies or other equivalents well known to those skilled in the art.
  • the present invention is based on the discovery of a new human goose lysozyme (LYG2) and a polynucleotide encoding LYG2, and the application of these components in the diagnosis, treatment or prevention of autoimmune inflammation, kidney, adrenal disease and cancer.
  • LYG2 a new human goose lysozyme
  • polynucleotide encoding LYG2 a polynucleotide encoding LYG2
  • the present invention includes a polypeptide LYG2 containing the amino acid sequence of SEQ ID NO: 1.
  • LYG2 has 182 amino acids and has four potential protein kinase C phosphorylation sites at residues T16, S26, T45 and T153.
  • LYG2 also has a potential signal sequence (residue M1 to residue S19 or so).
  • 2% Identity Both LYG2 and chicken goose-type lysozyme have four cysteine conserved in goose-type lysozyme. It is speculated that these cysteine form disulfide bonds at residues C39, C53, C62 and C92 of LYG2.
  • LYG2 contains aspartic acid residues conserved at the catalytic center of goose-type lysozyme at D117, and has a charged E105 residue and an acidic Q127 residue at the residue sites of the other two catalytic centers .
  • the invention also includes various variants of LYG2.
  • the preferred variant form of LYG2 is at least about 80%, more preferably at least about 90%, most preferably at least about 95% identical to LYG2 amino acid sequence, and contains at least one variant of LYG2 functional or structural characteristics.
  • the invention also includes a polynucleotide encoding LYG2.
  • the present invention includes a polynucleotide sequence including the sequence of SEQ ID NO: 2 which encodes LYG2.
  • the invention also includes variants of the polynucleotide sequence encoding LYG2.
  • a variant polynucleotide has at least about 70%, more preferably at least about 85%, and most preferably at least about 95% of the polynucleotide sequence identity to the polynucleotide sequence encoding LYG2.
  • a specific aspect of the present invention includes a variant of SEQ ID NO: 2 that has at least about 70%, more preferably about 85%, and most preferably at least about 95% polynucleotide sequence identity with SEQID NO. 2. .
  • Any one of the above variants of the polynucleotide can encode an amino acid sequence containing at least one functional or structural property of LYG2.
  • LYG2 codes may be generated.
  • the polynucleotide sequences some of which have low similarity to the polynucleotide sequences of known naturally occurring genes, the invention also includes a variant form of the polynucleotide sequence resulting from the selection of different codon combinations. These combinations are generated according to the naturally occurring polynucleotide sequence of LYG2 using the standard triplet genetic code.
  • LYG2 can also be encoded by basically different nucleotide sequences, such as:
  • the coding sequence contains non-naturally occurring codons; depending on the frequency with which the host uses a particular codon, codons can be selected to increase its presence in a particular prokaryotic or eukaryotic host The expression rate of the peptide.
  • Other reasons for wide-ranging changes in coding sequences include the production of RNA transcripts with more desirable properties, such as having a longer half-life than transcripts produced from natural sequences.
  • the present invention also includes a fragment that encodes a LYG2 derivative, or a fragment thereof, or is synthesized entirely by synthetic chemistry.
  • the synthetic sequence can be inserted into any of a number of available expression vectors and cell systems using reagents well known in the art.
  • mutations can be introduced into the sequence encoding LYG2 or any of its fragments by synthetic chemistry.
  • the washing step after hybridization can also vary in stringency.
  • the stringent conditions for washing can be defined by salt concentration and temperature. As mentioned above, the stringency of washing can be increased by reducing the salt concentration or increasing the temperature.
  • the stringent salt concentration of the washing step is preferably no higher than 30 mM NaCl and 3 mM trisodium citrate, and most preferably no less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • the stringent temperature conditions of the washing step are usually at least about 25 ° C, more preferably at least about 42 ° C, and most preferably at least about 68 ° (:.
  • it can be at 25 e C, 30 washing under the conditions of raM NaCl, 3mM trisodium citrate and 0.1% SDS.
  • the conditions can be 42, 15mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS.
  • the washing step is performed.
  • the washing step may be performed at 68 ° C, 15 mM NaCl, 1.5 ⁇ M trisodium citrate, and 0.1% SDS.
  • Various useful changes to these conditions are It is familiar to those skilled in the art.
  • sequencing preparation can be done automatically by thermal cycle control equipment and instruments, and sequencing can also be done automatically, for example using the ABI 377 sequencing system (PE Biosystems;). Sequences can be analyzed by computer programs and algorithms well known in the art (such as PCGENE, Clustal, etc.).
  • nucleic acid sequence encoding LYG2 can further extend the nucleic acid sequence encoding LYG2.
  • universal and nested primers can be used to amplify unknown sequences from genomic DNA cloned on a vector (see, eg, Sarkar, G. (1993) PCR Methods Applic, 2: 318-322).
  • reverse PCR uses primers that extend in different directions to amplify unknown sequences from a circular template.
  • the template is derived from restriction fragments containing known genomic loci and surrounding sequences (see, eg, Triglia, T. et al. (1988) Nucleic Acids Res.
  • the third method is capture PCR, which involves amplification DNA fragments in human and yeast artificial chromosomes adjacent to known sequences (see, eg, Lagerstrom, M. et al. (1991) PCR Methods Applic. ⁇ 111-119).
  • This method uses a number of restriction enzyme digestion and ligation methods to insert a double-stranded nucleotide sequence into a region of unknown sequence and then perform PCR. Other methods available for obtaining unknown sequences are known in the art. (See, for example, Parker, JD et al. (1991) NaCleic Acids Res. 19: 3055-3060).
  • genomic DNA walking with PCR and nested primers.
  • primer design can be performed using commercially available software, $ P 0LG04. 06 Primer AnaLysis Software (National Biosciences Inc. Madison, Minn.) Or other suitable program. It is 22-30 nucleotides, GC ratio is about 50% or higher, and can be annealed with the template at a temperature of about 68 fl C-72 e C.
  • a random primer library is more suitable because it often includes a sequence containing the 5 'region of the gene. Transcriptional regulatory regions.
  • capillary electrophoresis systems can be used to analyze or confirm the size or nucleotide sequence of sequencing or PCR products.
  • the capillary sequencing system which uses a flowable polymer for electrophoretic separation; four different fluorescent dyes to generate nucleotide-specific, laser-excited fluorescence; a charge-coupled camera device for detecting fluorescence emission The wavelength.
  • Use appropriate software such as: GENOTYPER and SEQUENCE NAVIGATOR analysis software (PE Biosystems) to convert the output / light intensity into electrical signals, and the entire process from sample loading to computer analysis and display of electronic data can be controlled by the computer.
  • Capillary electrophoresis is particularly suitable For sequencing a limited number of small cDNA fragments in a particular sample.
  • the polynucleotide sequence encoding LYG2 or a fragment thereof can be cloned in a recombinant DNA molecule, which can direct the expression of LYG2, or a fragment thereof, or a functional equivalent thereof in a suitable host cell. Due to the inherent degeneracy of the inherent genetic code, LYG2 can be expressed using other DNA sequences that encode substantially the same or functionally equivalent amino acid sequence as LYG2.
  • LYG2 amino acid sequence of LYG2 or any part thereof can be produced by directed synthesis and / or fusion with any part thereof from other proteins to produce a variant polypeptide.
  • the peptide can be substantially purified by preparative high performance liquid chromatography. (See, RM Chiez and FZ Regrier (1990) Methods Enzymol. 182: 392-421) 0
  • the composition of synthetic peptides can be confirmed by amino acid analysis or sequencing (see, eg, T. Creighton (1984) Proteins, structures and Molecular Properties, WH Freeman and CO., New York, NY).
  • a nucleotide sequence encoding LYG2 and its derivatives is inserted into a suitable expression vector, for example, one that contains the necessary transcription and translation control of the inserted coding sequence in a suitable host.
  • a suitable expression vector for example, one that contains the necessary transcription and translation control of the inserted coding sequence in a suitable host.
  • Vector of elements include, for example, enhancers, constitutive and inducible promoters, vectors, and the 5 'and 3' untranslated regions of the polynucleotide sequence encoding LYG2. These elements differ in their regulatory capabilities and specificity.
  • the specific start signal enables more efficient translation of the sequence encoding LYG2.
  • Such signals include the ATG start code and its adjacent sequences.
  • Such as the K0ZAK sequence provided that the sequence encoding the LYG2 and its start codon and upstream regulatory sequences are inserted into a suitable expression vector, no other transcription or translation control signals are required. However, if only the coding sequence or one of its fragments is inserted into the vector, the foreign signal including the ATG start codon in the box must be provided by the vector. Exogenous translation elements and start codons can come from different sources-either natural or synthetic. Containing enhancers suitable for use in a particular host cell system can increase expression efficiency. (See, eg, D. Scharf et al. (1994) Results Probl. Cell Differ. 20: 125-162).
  • LYG2 coding sequences can be packaged and expressed using different expression vector / host systems. They include, but are not limited to, microorganisms, such as those that transform recombinant bacteriophages, plasmids, or cosmid DNA expression vectors; yeasts that transform yeast expression vectors; insect cell systems infected with viral expression vectors (such as: baculovirus); Viral expression vector or bracketed bacterial expression vector (such as Ti material or pBR332 plasmid), (such as: cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) transformed plant cell system; or animal cell body.
  • the invention is not limited by the host cell used.
  • cloning and expression vectors in bacterial systems based on the intended use of the polynucleotide sequence encoding LYG2.
  • conventional cloning, subcloning, and proliferation of a polynucleotide sequence encoding LYG2 can be performed using a multifunctional E. coli vector such as PBLUESCRIPT phagemid (Strat LEGO) or PSPORT2 plasmid (Life Technologies). Because the connection of the LYG2 coding sequence to the multicloning site of the vector will destroy the LacZ gene, this makes it possible to perform colorimetric screening of transformed bacteria containing recombinant molecules.
  • these vectors are useful for in vivo transcription, dideoxy sequencing, single-strand rescue in conjunction with helper phages, and for creating nested deletions in cloned sequences (see, eg, Van rleeke, G., and SW Schuster (1989) JB Chem. 264: 5535-5509).
  • LYG2 When a large amount of LYG2 is required, for example, for the production of antibodies, it is necessary to use LYG2 to target high-level expression vectors.
  • a vector containing a strongly inducible T5 or T7 phage promoter can be used.
  • Yeast expression systems can be used to produce LYG2.
  • Many vectors containing constitutive or inducible promoters can be used in Saccharomyces cerevisiae or pichia Pastors.
  • these vectors can direct the secretion or retention of expression proteins, and can integrate foreign sequences into the host's genome to stabilize proliferation.
  • Plant systems can also be used to express LYG2. Transcription of the LYG2 coding sequence can be performed by viral promoters, such as the Camvl9S and 35s promoters used alone or in combination with the TWV ⁇ leader sequence (N. Takamatsn (1984) S224: 838-834, and J. Winter et al. (1191) results probl. Cell differ. 17: 85-105); or plant promoters such as the small subunit of RUBISC0 or the promoter of heat shock protein (CoruLYG2i, G. et al. (1984) EMB0 J. 3: 1671- 1680; Broglie, R. et al. (1984) Science 224: 838-843; and Winter, J.
  • viral promoters such as the Camvl9S and 35s promoters used alone or in combination with the TWV ⁇ leader sequence (N. Takamatsn (1984) S224: 838-834, and J. Winter et al. (1191)
  • the LYG2 coding sequence can be linked to an adenovirus transcription I translation complex consisting of a late promoter and a triplet leader sequence.
  • the insertion of non-essential E1 or E3 regions in the viral genome can be used to obtain infectious viruses that express LYG2 in host cells (see, eg, J. Logan and T. Shenk (1984) Proc. Natl. Acad. Scio81: 3655-3659 ).
  • transcription enhancers such as the Lloyd's intratumor virus (RSV) enhancer, can be used to improve Expression in host cells.
  • RSV Lloyd's intratumor virus
  • HACS Artificial chromosomes
  • plasmids By conventional delivery methods (liposomes, polycationic amino polymers, or vesicles), about 6Kb-10Mb of HACs can be constructed and delivered for therapeutic purposes. (See, eg, JT Harringtoo et al. (1997) Nat. Genet. 15: 345-355).
  • LYG2 For long-term production of recombinant proteins in mammalian systems, LYG2 must first be stably expressed in cell lines.
  • an LYG2 encoding gene can be transformed into a cell line using an expression vector containing a virus origin of replication and / or endogenous expression elements and a selectable marker gene located on the same vector or different vectors. After introduction of the vector, cells can be allowed to grow on the enriched medium for about 1-2 days before being transferred to the selection medium.
  • the purpose of the selectable marker is to confer resistance to the selective agent, and its presence allows cells that successfully express the introduced sequence to grow and recover.
  • tissue culture techniques allows proliferation of resistant clones of stably transformed cells.
  • the presence or absence of marker gene expression suggests that the gene we are interested in also exists, the existence and expression of this gene need to be confirmed.
  • the loss of function of the marker gene can be used to identify transformed cells containing the LYG2 coding sequence.
  • the marker gene is placed in tandem with the LYG2 coding sequence and under the control of a single promoter, the expression of the marker gene under the action of induction or selection usually also shows expression on a tandem basis.
  • host cells containing and expressing LYG2 can be identified using a variety of method steps known to those skilled in the art. These method steps include, but are not limited to, DNA-DNA or DNA-RNA hybridization, PCR amplification, and assay methods including various methods for detecting nucleic acid and protein sequences based on membranes, solutions, and chips. Either specific polyclonal or monoclonal antibodies are used to detect and measure LYG2 expression, and these immunological methods are known in the art. Examples of such technologies include enzyme-linked immunosorbent assays (ELISAS), radioimmunoassays (RIAS), and fluorescence-activated cell sorting (FACS).
  • ELISAS enzyme-linked immunosorbent assays
  • RIAS radioimmunoassays
  • FACS fluorescence-activated cell sorting
  • a monoclonal-based two-site immunoassay using monoclonal antibodies that are active on two independent epitopes on LYG2 is preferred, but a competitive binding assay can also be used.
  • assays are well known in the art (see, eg, R. Hampton et al. (1990) serological methods, a laboratory manual, APS Press, stpaul minn. Part IV; JZ col igan et al. ( 1997, and regular supplements) Current Protocols in ImMmnology, Greene Pub. Associates and wiley-interscience, New York, Ya .; and 0.5. 1 ⁇ 0 ⁇ 0 et al. (1983) 1 1.6. Med. 158 : 1211-1216)
  • Methods for generating labeled hybridization probes and PCR probes to detect sequences related to a polynucleotide encoding LYG2 include oligonucleotide labeling, nick translation, end labeling or PCR amplification with labeled nucleotides Alternatively, the sequence encoding LYG2 or any of its fragments can be cloned into a vector to produce an mRNA probe. Such vectors are known in the art and are commercially available.
  • RNA polymerases such as T7, T3 or SP6 and labeled nucleotides.
  • T7, T3 or SP6 RNA polymerases
  • Appropriate reporter molecules and labels can facilitate the detection. These molecules and labels include radioisotopes, enzymes, fluorescent agents, chemiluminescent agents or chromophores, but also substrates, cofactors, inhibitors, magnetic particles, and similar Thing.
  • a host cell transformed with a nucleotide sequence encoding LYG2 can be transformed into cells suitable for protein expression and recovery from cell culture. Culture under the condition of protein harvesting. Whether the protein produced by the transformed cell is secreted or stays inside the cell depends on the sequence and / or vector used.
  • an expression vector containing a polynucleotide encoding LYG2 may be designed to include a signal sequence that directs the secretion of LYG2 through a prokaryotic or eukaryotic cell membrane.
  • the selection of the host cell line can be based on the cell line's ability to regulate or modify the expression of the inserted sequence.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing of protein precursors can define protein targeting, folding, and / or activity.
  • Different host cells with specific protein post-translational processing capabilities or mechanisms can be obtained from American Type Culture Collection (ATCC, Bathesda, Md). These host cells can be selected to ensure Correct processing and modification of foreign proteins.
  • a natural, modified, or recombinant nucleic acid sequence encoding LYG2 can be linked to a heterologous sequence, and the fusion protein can be expressed in any of the above-mentioned host systems.
  • a chimeric LYG2 protein containing a heterologous component that can be recognized by a commercially available antibody can help screen a peptide library to obtain an inhibitor that inhibits LYG2 activity.
  • Heterologous protein components and polypeptide components can also facilitate the purification of fusion proteins using commercially available affinity matrices.
  • Such components include, but are not limited to, glutathione S-transferase (GST), maltose-binding protein (MBP), thioredoxin (Trx), calmodulin-binding peptide (CBP), 6-His, FLAG , C- MYC and hemagglutinin (HA;).
  • GST, MBP, TRX, CBP, and 6-His can purify the corresponding fusion proteins on immobilized glutathione, maltose, phenylene oxide, calmodulin resin, and metal chelate resin, respectively.
  • the fusion protein can be constructed artificially so that it contains a protein cleavage site between the LYG2 coding sequence and the heterologous protein sequence, so that LYG2 can be separated from the heterologous components after purification.
  • radiolabeled LYG2 in vitro synthesis can be completed.
  • These systems couple the transcription and translation of protein coding sequences of the T7, T3 or SP6 promoters. Translation occurs under conditions of a radiolabeled amino acid precursor, preferably Sup 35 S-methionamine.
  • LYG2 fragments can be produced not only from recombinant products, but also directly from direct peptide synthesis in solid-phase synthesis techniques (see, eg, Creighton, supra, pp55-60). Protein synthesis can be performed by manual (operational) technology or automated technology. For example, the ABI msdel 43 / A peptide synthesizer (PE Biosystems) can be used to automate protein synthesis. Various fragments of LYG2 can be synthesized separately and then combined to produce the full-length molecule of LYG2.
  • LYG2 There are chemical and structural similarities between LYG2 and human-derived goose-type lysozyme LYG2.
  • LYG2 is expressed in cancerous, inflamed, renal, breast, adrenal, colon, and neural tissues. Therefore, LYG2 mainly plays a role in autoimmune inflammation, diseases of the kidney and adrenal glands, and cancer.
  • LYG2 or a fragment or derivative thereof can be administered to a subject to treat or prevent an autoimmune I inflammatory disease.
  • This type of autoimmune inflammatory disease includes (but is not limited to): acquired immunodeficiency syndrome (AIDS), Addison's disease (chronic adrenal insufficiency), adult respiratory disorder, allergies, joint rigid spine Inflammation, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, beriberi, cholecystitis, contact dermatitis, localized ileitis, allergic dermatitis, bronchitis, diabetes , Emphysema, staged lymphocytopenia and lymphogranuloma, bone marrow erythrocytosis, erythema, atrophic gastritis, angiocystic nephritis, pulmonary hemorrhagic nephritis syndrome, gout, exophthalmic goiter
  • a vector capable of expressing LYG2 or a fragment or derivative thereof can be administered to a subject to treat or prevent autoimmune / inflammatory diseases, including, but not limited to, those diseases described above.
  • a pharmaceutical composition containing substantially pure LYG2 can be administered to a subject together with a suitable pharmaceutical carrier to treat or prevent an autoimmune / inflammatory disease.
  • a suitable pharmaceutical carrier to treat or prevent an autoimmune / inflammatory disease.
  • an agonist that modulates LYG2 activity can be administered to a subject to treat or prevent diseases of autoimmune inflammation, including, but not limited to, the diseases listed above.
  • kidney diseases include (but are not limited to): renal amyloidosis, renal hypertension; aldosteronism; Addison's disease; renal failure; glomerulonephritis; chronic glomerulonephritis; Renal cyst disease and developmental abnormalities, such as polycystic disease, renal dysplasia and cortical or medullary sac, hereditary nephropathy (PRD), such as: trapped and autosomal-dominated PRO: medullary cystic disease; Renal cavernous medulla and tube dysplasia; Alprot's syndrome; non-renal cancers that affect the physiological function of the kidney, such as: bronchiomas, or basal tumors of the brain; a variety of myeloma; renal adenocarcinoma; By digestion, injection, inhalation or absorption of any medicament, chemical or biological agent, such
  • a vector capable of expressing LYG2 or a fragment or derivative thereof can be administered to a subject to treat or prevent kidney diseases, including, but not limited to, those diseases described above.
  • a pharmaceutical composition containing substantially pure LYG2 may be administered to a subject together with a suitable pharmaceutical carrier to treat or prevent kidney disease, which includes, but is not limited to, the diseases provided above.
  • an agonist that modulates LYG2 activity can be administered to a subject to treat or prevent kidney disease, which includes, but is not limited to, the diseases listed above.
  • LYG2 or a fragment or derivative thereof can be administered to a subject to treat or prevent adrenal disease.
  • adrenal diseases include (but are not limited to): adrenal hyperplasia, cancer or adenoma, alkalosis-related hypertension, amyloidosis, hypokalemia, Cushing's disease, liddle syndrome, and Arnold- Healy- Gorden syndrome, pheochromocytoma, and Addison's disease.
  • a vector capable of expressing LYG2 or a fragment or a derivative thereof can be administered to a subject to treat or prevent an adrenal disease, which includes, but is not limited to, the diseases described above.
  • a pharmaceutical composition containing basic LYG2 can be administered to a subject together with a suitable pharmaceutical carrier to treat or prevent adrenal diseases, which include (but are not limited to) the diseases provided above.
  • an agonist that modulates LYG2 activity can be administered to a subject to treat or prevent adrenal diseases, including, but not limited to, the diseases listed above.
  • an antagonist of LYG2 can be administered to a subject to treat or prevent cancer.
  • cancers include (but are not limited to): adenoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratoma, and especially It includes: adrenal cancer, bladder cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, cervical cancer, gallbladder cancer, nerve center cancer, gastrointestinal cancer, heart cancer, kidney cancer, liver cancer, lung cancer, muscle cancer, Ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, prostate cancer, salivary cancer, skin cancer, spleen cancer, testicular cancer, thymic cancer, thyroid cancer, and uterine cancer.
  • an antibody that specifically binds LYG2 can be used directly as an antagonist or indirectly as a targeting mechanism or delivery mechanism for bringing Agent 5 into cells or tissues expressing LYG2.
  • a vector expressing a complementary strand of a LYG2 encoding polynucleotide may be administered to a subject.
  • cancers include (but are not limited to) the cancers described above.
  • any one of the proteins, antagonists, antibodies, agonists, complementary sequences or vectors of the invention can be administered in conjunction with other suitable therapeutic agents.
  • suitable therapeutic agents According to conventional pharmacological principles, one of ordinary skill in the art will be able to complete the selection of a suitable agent for combination therapy.
  • the combination of different therapeutic agents can synergistically affect the treatment and prevention of the various diseases mentioned above. In this way, people can achieve therapeutic efficacy with fewer doses of various drugs, thereby reducing the possibility of adverse side effects.
  • Antagonists of LYG2 can be produced using methods commonly known in the art.
  • purified LYG2 can be used to generate antibodies or to screen a library of agents to identify agents that specifically bind to LYG2.
  • Anti-15 antibodies against LYG2 can also be generated using methods well known in the art.
  • Such antibodies include (but are not limited to): polyclonal antibodies; monoclonal antibodies, chimeric antibodies and single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Neutralizing antibodies eg, neutralizing antibodies that inhibit the formation of dimers are particularly suitable for treatment.
  • LYG2 or any of its immunogenic fragments or oligopeptides can be injected to immunize various hosts, including goats, rabbits, rats, mice, humans, and other hosts. For lower> 0 applications involving the production of monoclonal antibodies, rats and mice are the preferred hosts.
  • various adjuvants can be used depending on the host. Such adjuvants include (but are not limited to): Freimd's adjuvant; mineral gels such as aluminum hydroxide; surface-active substances such as lysolecithin; polycations; oil emulsions; keyhole limpet hemocyanin (KLH); And dinitrobenzoic acid.
  • BCG human adjuvants
  • BCG BCG
  • BCG BCG
  • Antibodies A Laboratory Manual, cold spring 15 hartor Laboratore, cold spring harbor, N. Y.
  • the oligopeptide, peptide or fragment used to induce the production of an antibody against LYG2 has an amino acid sequence consisting of at least about 5 amino acids, more preferably, at least about 14 amino acids.
  • these oligopeptides or fragments thereof are identical to a part of the amino acid sequence of the natural protein, and contain the complete amino acid sequence of a naturally occurring small molecule.
  • the short LYG2 amino acid fragment can be fused to another protein such as the short amino acid fragment of KLH, and can be used to generate antibodies against chimeric molecules.
  • Monoclonal antibodies to LYG2 can be prepared by the technique of culturing serial cell lines to produce antibody molecules. These technologies include (but are not limited to): hybridoma technology, human B-cell hybridoma technique, and EBV hybridoma technology. (See, G. Koller et al. (1975) Nature256: 495-497: D. Kozbor et al. (1985) J. Immunol. Methodsgl: 31-42; RJ cote et al. (1983) proc. Natl. Acad. Sci. 80: 2026-2030; '5 and S. p. Cole et al. (1984) Mol. Cell Biol. 62: 109-120).
  • chimeras can be utilized, such as splicing mouse antibody genes to human antibody genes to obtain molecules with appropriate antigen specificity and biological activity.
  • splicing mouse antibody genes to human antibody genes to obtain molecules with appropriate antigen specificity and biological activity.
  • the LYG2-specific single-chain antibody can be generated using the above-mentioned technology for producing single-chain antibodies by using methods known in the art. By random shuffling of chains of immunoglobulin libraries, different idiotype antibodies with related specificity can be generated (see, eg, DR Burton (1991) proc, nal. Acad. Sci. 88: 10134-10137).
  • Antibodies can be induced in the lymphocyte population in vivo. Alternatively, as disclosed in the literature, antibodies can be generated by screening immunoglobulin libraries or a series of highly specific binding reagents. (See, R. Orlandi et al. (1989) proc, nal. Acad. Sci. 88: 3833-3837; and G. Winter et al. (1991) nature349: 293-299)
  • Antibody fragments containing LYG2 specific binding sites can also be generated. Such fragments include (but are not limited to); F (ab) ' 2 fragments produced by digesting antibody molecules with pepsin, and Fab fragments produced by reducing the disulfide bridge of the F (ab)' 2 fragment. Alternatively, construct Fab expression libraries to quickly and easily identify monoclonals with the desired specificity
  • immunoassays can be used for antibody screening to identify antibodies with the desired specificity and minimal cross-reactivity.
  • immunoassay protocols in the art. These protocols use monoclonal or doxlon antibodies for competitive binding or immunoradiation analysis. It usually includes the measurement of the complex formed by LYG2 and its specific antibody, and a monoclonal, two-site-based immunoassay using a monoclonal antibody that is reactive with two non-interfering LYG2 epitopes is more preferable But competitive binding analysis can also be applied.
  • a polyclonal antibody preparation should contain at least 1-2 mg of specific antibody / ml, and preferably 5-10 mg / ml, in order to be suitable for experimental operations that require LYG2-antibody complex precipitation reaction.
  • Methods for measuring antibody specificity, titer, affinity, and descriptions of antibody properties and uses in various applications are often readily available (see e. G. Catty, supra and doligan et al. Supra) o
  • a polynucleotide encoding LYG2, or a fragment thereof, or a complementary sequence thereof can be used for treatment.
  • the complementary sequence of a polynucleotide encoding LYG2 can be used to block mRNA transcription.
  • cells can be transformed by the complementary sequence of a polynucleotide encoding GOLY. Therefore, complementary molecules or complementary fragments can be used to regulate LYG2 activity or to regulate gene function.
  • Oligonucleotides or large fragments or their antisense sequences can be designed by various sites on the coding region or control region on the LYG2 coding sequence.
  • Various expression vectors derived from retrovirus, adenovirus, herpes virus, pox virus, or various bacterial plasmids can be used to transfer the nucleotide sequence into the target organ, target tissue or cell population.
  • Various techniques well known to those skilled in the art can be used to construct vectors to express complementary sequences of the LYG2 coding sequence (see, eg, sambrook supra, and Ausubel, supra). 0
  • the gene encoding LYG2 or a fragment thereof can be turned off.
  • This type of construct can be used to mediate the entry of untranslated or antisense sequences into a cell. Even in the absence of integration with DNA, these vectors can continue to transcribe RNA molecules until they are inactivated by endogenous nucleases. In the case where the vector cannot be replicated, transient expression can last for one month or longer, such as in the case where the vector system has a suitable replication factor, and the duration can be further extended.
  • the expression gene can be modified. Derived from the oligo at the transcription start site Nucleotides such as those derived from the -10 to +10 region are preferred. Similarly: Inhibition can be produced by triple-helix base-pairing. It is very useful because it prevents the double helix from being fully melted, thereby inhibiting the binding of DNA to polymerases, transcription factors, or regulatory molecules.
  • Ribozymes RNA molecules with enzymatic activity, can also be used to catalyze the cleavage of RNA-specific cleavage sites.
  • the mechanism of a ribozyme reaction involves the specific binding of a ribozyme molecule to a complementary target RNA, followed by cleavage of the target RNA.
  • a synthetic hammer-type fancy ribozyme molecule can specifically and efficiently catalyze the cleavage of the LYG2 coding sequence.
  • Any potential specific ribozyme action sites on the target RNA can be detected initially by scanning the target molecule. These sites include the following sequences: GUA, GUU, and GUC.
  • RNA sequence 15 ⁇ 20bp
  • the suitability of candidate targets can also be determined by testing RNase protection for hybridization with complementary oligonucleotides.
  • the complementary ribonucleic acid molecules and ribozymes in the present invention can be prepared by any method for synthesizing nucleic acid molecules known in the art. These techniques include chemical synthesis, such as solid-phase phosphoramidite solid-phase synthesis.
  • RNA molecules can be produced by in vitro transcription or in vivo transcription of the coding sequence of LYG2. These DNA sequences can be cloned into a large class of vectors with suitable promoters such as the T7 or S p6 RNA polymerase promoter.
  • a cDNA construct that synthesizes complementary RNA constitutively or inducibly can be introduced into a cell line, cell, or tissue.
  • RNA molecules can be modified to increase their intracellular stability and half-life. Possible modifications include (but are not limited to) adding a flanking sequence to the 5 'and, or 3' terminus of the molecule, or replacing the phosphodiester between the molecular backbone with a phosphorothioate bond or a 2'0-methyl bond. Key to connect. These modified forms are also applicable to the preparation of PNAs and can be extended to other nucleotide molecules, including non-common bases such as inosine, but also acylated, methylated, sulfurized or other similar modified forms of thymine, cytosine Pyrimidine, guanine, adenine and uracil. These modified forms are not easily recognized by endogenous endonucleases.
  • vectors into cells or tissues There are many ways to make vectors into cells or tissues, and they are equally suitable for in vivo and in vitro therapy.
  • the vector is first introduced into the stem cells taken from the patient, then the germ cells are cloned, and then transferred back to the original patient through autologous transplantation.
  • the above methods can be used to introduce vectors by transfection, lymphatic injection, or polycationic amino polymers (Goldamn. C. K. et al. 1997. Nature Biotechnology 15: 462-466). It can be used for any subject in need of this therapy, including: mammals such as dogs, cats, cows, sheep, rabbits, monkeys, and most preferably humans.
  • a pharmaceutical component or a sterile component in combination with a drug-loadable carrier to achieve the above-mentioned therapeutic effect.
  • These pharmaceutical components include LYG2, antibodies to LYG2, and mimics, stimulants, antagonists, and inhibitors of LYG2.
  • These components can be used alone or in combination with at least one other agent.
  • a stable compound that can be used in any sterilized and can be present in the body, including (but not limited to) saline, buffered saline, dextrose, and the like.
  • saline buffered saline
  • dextrose dextrose
  • the pharmaceutical composition of the present invention can be administered by any route. These include (but are not limited to): oral, intravenous, intramuscular, intraarterial, bone marrow, subcutaneous or peritoneal.
  • these pharmaceutical ingredients also include suitable drug-bearing carriers containing stimulants and adjuvants, which help turn the active complex into a usable pharmaceutical ingredient.
  • suitable drug-bearing carriers containing stimulants and adjuvants which help turn the active complex into a usable pharmaceutical ingredient.
  • compositions for oral administration can be carried with a variety of pharmaceutical carriers suitable for oral administration known to those of ordinary skill in the art. These medicinal ingredients are in the form of tablets, pills, dragees, capsules, liquids, suspensions, etc. for patients to absorb.
  • the active ingredient can be mixed and ground with a solid solid to form a granular mixture, and then made into a tablet or sugar pill core, and an appropriate adjuvant can be added.
  • Suitable solids include sugar and protein fillers, including lactose, sucrose, mannitol, sorbase, starch extracted from corn, wheat, rice, potatoes, or other plants, and cellulose such as methylated cellulose. Or-fiber or methyl sodium cellulose, gums and proteins such as gelatin, collagen. If necessary, dispersants and solubilizers can also be added, such as agar, alginic acid and its salts, such as solid alginates.
  • the core of the pill must be combined with a suitable coating, such as a saturated sugar solution, (may also contain gum arabic, polyvinylpyrione, polyethylene glycol and / or neodymium dioxide) and a suitable organic or mixed solvent, Can be in tablets or colors to distinguish the product or indicate the amount of active ingredients, such as dosage forms.
  • a suitable coating such as a saturated sugar solution, (may also contain gum arabic, polyvinylpyrione, polyethylene glycol and / or neodymium dioxide) and a suitable organic or mixed solvent, Can be in tablets or colors to distinguish the product or indicate the amount of active ingredients, such as dosage forms.
  • Orally-available pharmaceutical products include fit-type capsules made of gelatin and soft, sealed capsules made of gelatin and three types of color coats of glucosidase and sorbase.
  • fitted capsules also contain filling and binding (such as lactose or starch), lubricants (such as talcum powder or magnesium stearate), and stabilizers.
  • the active ingredient is dissolved or suspended in a suitable liquid.
  • a suitable liquid such as grease, liquid or polyethylene glycol liquid, stabilizers can be added to them.
  • the pharmaceutical dosage form for injection can be formulated in an aqueous solution, more preferably a physiologically compatible buffer such as Hanks' solution or physiological saline.
  • the injection can contain substances which increase the viscosity of the suspension such as sodium carboxymethylcellulose, sorbitol or dextran.
  • the active complex suspension can also be made into a suspension injection of the lipid type.
  • Suitable lipophilic solvents or vehicles include fats, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers can also be used as vehicles.
  • suspensions may also contain suitable stabilizers or agents that increase the solubility of the complexes to prepare highly concentrated solutions.
  • Formulations for topical or nasal administration contain osmotic agents that are commonly known in the art to penetrate specific barriers.
  • the pharmaceutical ingredients in the present invention can be produced by methods known in the art: such as conventional mixing, dissolving, granulating, pelleting, milling, emulsifying, encapsulating or lyophilizing processes.
  • salts can be combined with various acids to form salts, which are stored in the form of salts.
  • acids include (but are not limited to): hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, and succinic acid. Salts are more soluble in aqueous solutions or other protic solvents than the corresponding basic ingredients.
  • a better manufactured product is in the form of a lyophilized powder, which may include some or all of the following ingredients, 1mm-50mm histidine, 0.1% to 0.2% sucrose, 2% gc 7% Mannitol, pH range is 4.5 to 5.5, and they are mixed with buffer before use.
  • an antibody that specifically binds to LYG2 can be used in the diagnosis of a disease characterized by LYG2, or for monitoring analysis of patients treated with LYG2 or an activator, antagonist or inhibitor of LYG2.
  • Antibodies for diagnostic purposes can be prepared in the same manner as described above.
  • the diagnostic analysis of LYG2 includes: using the antibody and label to detect LYG2 in human fluid cells or tissue extracts.
  • the antibody may be unmodified, or it may be labeled covalently or non-covalently with a reporter molecule. There are many reporters available in the field, several of them As mentioned above.
  • LYG2 expression values can be determined by combining body fluids or cell extracts from normal experimental animal subjects (preferably humans) with LYG2 antibodies under conditions suitable for complex formation.
  • the standard compound content can be quantitatively determined by various methods, and more preferably optical measurement methods.
  • the measured values of LYG2 expression in diseased samples of experimental subjects, controls, and living tissues are compared with standard values, and the differences between the two can be used to determine parameters for disease diagnosis.
  • polynucleotides encoding LYG2 can be used for diagnostic purposes.
  • Such polynucleotides include: oligonucleotide sequences, complementary RNA, DNA molecules, and PNA molecules. These polynucleotides can be used for Detection and quantitative determination of gene expression in living tissues, among which LYG2 expression may be related to disease. Diagnostic analysis can be used to determine the loss, presence, and excess of LYG2 expression, and to monitor the level of adjustment of LYG2 during treatment.
  • the probe preferably comprises a sequence consisting of 10-846 consecutive nucleotides in SEQ ID No. 2 or the sequence of its complementary strand.
  • the probe can be used for the detection of related sequences.
  • the probe has at least 50% sequence identity with any LYG2 coding sequence.
  • the hybridization probe of the present invention can be DNA or RNA.
  • Methods for preparing specific probes for DNA encoding LYG2 include: cloning a polynucleotide encoding LYG2 or a LYG2 derivative into a vector for preparing an mRNA probe, these vectors are known in the art, commercially available, and It can be used to synthesize RNA probes in vitro by adding appropriate RNA polymerase and appropriate labeled nucleotides.
  • Hybridization probes can be labeled with various reporter genes, such as radionucleosides, such as 32 P or 35 S, and enzyme labels, such as alkaline phosphatase coupled to the probe via an avidin / biotin coupling system, And other similar methods.
  • Polynucleotides encoding LYG2 are useful in the diagnosis of diseases associated with LYG2 expression. These diseases include (but are not limited to): autoimmune / inflammatory diseases such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult dyspnea syndrome, allergies, arthritis spondylitis, amyloidosis, anemia, Asthma, atherosclerosis, 'autoimmune hemolytic anemia, autoimmune thyroiditis, beriberi, cholecystitis, contact dermatitis, Crohn's disease, allergic dermatitis, bronchitis, diabetes, emphysema stage lymphocytopenia And lymphogranuloma, bone marrow erythrocytosis, erythema, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Grave's disease, acute enteritis, sclerosis, severe myasthenia, myocarditis,
  • Polynucleotide sequences encoding LYG2 can be used in Southern or Northern analysis, or other membrane-based analysis, for PCR technology; measuring rods, needle tips, and ELISA-based analysis; and micromatrix analysis for patient fluids or tissues to detect LYG2 expression The change. These qualitative or quantitative analysis methods are well known in the art.
  • the nucleotide sequence encoding LYG2 can be labeled by conventional methods and then added to a patient's body fluid or tissue sample under conditions suitable for the formation of a hybridization complex. After warming, the samples were washed, and the signal values were quantitatively measured and compared with the standard values. If the signal value in the patient sample significantly changed compared to the control group, the change in the level of the nucleotide encoding LYG2 indicates the existence of the relevant lesion.
  • These analyses can be used in animal research and clinical trials to evaluate the efficacy of a particular therapy. It can also be used to monitor the treatment of a case.
  • a map of normal or standard LYG2 expression must be determined, which can be obtained by extracting body fluids or cell extracts from normal subjects of animals or human Under conditions suitable for hybridization or amplification, it is mixed with the sequence encoding LYG2 or a fragment thereof.
  • a standard hybrid number can be obtained. The standard value obtained in this way can be compared with the Comparison of the values of the samples presented by patients with symptoms, the magnitude of the deviation can be used to determine the presence of lesions.
  • the hybridization analysis can be repeated periodically to determine if the expression level in the patient is starting to approach the level observed in normal individuals. Continuous analysis results can be used to show the efficacy of the treatment over a period of time. From days to months.
  • oligonucleotides designed based on the LYG2 coding sequence involve the use of PCR. These oligomers can be produced by chemical synthesis, enzymatic digestion, or in vitro. The oligomers preferably contain fragments of the LYG2 coding sequence, or Complementary fragments can be used under optimized conditions to identify specific genes or conditions. Oligomers can also be used to detect or quantitatively measure similar DNA or RNA sequences under low stringency conditions.
  • Methods that can be used to quantify LYG2 expression include: radiolabeled or biotinylated nucleotides, co-amplification of control nucleic acids, and derivation based on standard curves (see eg raelby, dc et al. (1993) J. Imaunol. Methods 159 : 235-244; and Duplaa, C. et al. (1993) Anal. BiocheM 212: 224-236), the quantitative analysis of multiple samples can be quickly obtained by ELISA-like analysis. In this method, oligomers are diluted to different concentrations and can be quickly quantified by spectrophotometric calorimetry.
  • the oligonucleotide or its full-length fragment obtained from the polynucleotide sequence described above can be used as the object of a micromatrix, and the micromatrix column can be used to simultaneously detect the expression levels of a large number of genes, and Can detect gene mutations, mutations and polymorphisms. This information can be used to infer gene function, understand the genetic basis of the disease, diagnose the disease, and improve and monitor the activity of agents.
  • Microarray methods can be prepared, applied, and analyzed by means known in the art (see eg Brennan, TM et al. (1995) US Pat. No. 5, 474, 796; Schena, M. et al (1996) Proc Natl, Acad Sci, 93: 10614-10619) PCT appvication wo95 / 35505, Heller, RA et al. (1997) Prol. Natl. Acad. Sci. 94: 2510-2155; and Heller, MJ et al. (1197 ) U. S Pat No. 5, 605, 622).
  • the nucleic acid sequence encoding LYG2 can be used to generate probes useful for locating natural genomic sequences.
  • the sequence can be located on a certain chromosome, a specific region of a chromosome, or an artificial chromosome.
  • Materials such as human artificial chromosomes (HALs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BALs), bacterial PI constructs, or single-chromosome cDNA libraries (see eg Price, L, M. (1993) Blood Rev. 7: 127- 134; and Trask, BJ (1191) Trends Genet. 7: 149-154) 0
  • Fluorescence in situ hybridization can be combined with other physical mapping techniques and genetic mapping data (see eg Heinz-Ulrich et al. (1995) in Meyers, RA led) Molecular Biology and Biotechnology, VCH Publishers New York, N. T, PP965-968 ). Examples of genetic map data can be found in various scientific manuals or OMIM sites.
  • the relationship between the physical map of the LYG2 coding gene and the susceptibility to a particular disease can be used to determine the location of the DNA associated with the disease
  • the nucleotide sequence of the present invention can be used to detect differences in gene sequences between normal persons, carriers and patients with disease.
  • chromosome in situ hybridization preparations and physical mapping techniques such as the use of lasso analysis to determine chromosome markers
  • it can be used to further expand the gene map.
  • the layout of a gene on the chromosome of another mammal can provide information about related markers even when the human chromosome is located or the long and short arms are unknown.
  • the new sequence can be located on the chromosome through a physical map. This provides useful information for disease-locating workers using localized cloning or other gene-finding technologies.
  • any The sequence located in this region may be a disease-related gene or a regulatory gene, which can be further studied (see eg, Gatti, R. A et al. (1988) Nature 336: 577-580).
  • the nucleotide sequence of the present invention It can also be used to detect differences in chromosome positions based on translocations and insertions in normal, carrier, or disease patients.
  • a catalytic fragment or an immunogenic fragment of LYG2, or an oligopeptide can be used to screen a library of complexes (by various drug screening techniques).
  • the fragments used in this method can be soluble in the liquid phase, localized on a solid support, localized on the cell surface, or localized intracellularly. The formation of the linker complex between LYG2 and the drug being tested is measurable.
  • Another drug screening technique provides an efficient screening method for identifying complexes that bind to the protein of interest (see e. G. Geysen et al. (1984) PCT application W084103564).
  • a small amount of many different test compounds are synthesized on the surface of solid materials, such as plastic needles or other surfaces. These test substances can react with LYG2 or its fragments. After the reaction, the bound LYG2 can pass through
  • purified LYG2 can also be directly wrapped on a tray for use in the aforementioned drug screening technology.
  • non-neutralizing antibodies can be used to bind the peptide and immobilize it on a solid support.
  • a competitive drug screening analysis can be used. This method uses a neutralizing antibody that specifically binds to LYG2 to compete with the test complex for binding to LYG2. In this method, antibodies can be used to detect any peptide that has one or more LYG2 antigenic determinants.
  • nucleotide sequence encoding LYG2 is used in any molecular biology technology that has been developed. As long as the new technology is based on the properties of known nucleotides, these characteristics include (but are not limited to): gene coding Triad and base-specific pairing.
  • the full-length human LYG2 nucleotide sequence or a fragment thereof of the present invention can usually be obtained by a PCR amplification method, a recombinant method, or a synthetic method.
  • primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available cDNA library or cDNA prepared according to conventional methods known to those skilled in the art can be used.
  • the library is used as a template and the relevant sequences are amplified. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then stitch the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually cloned into a vector, and then transferred into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • relevant methods can also be synthesized by artificial synthesis, especially when the fragment length is short.
  • long sequences can be obtained by synthesizing multiple small fragments first and then ligating them.
  • the DNA sequence encoding the protein (or a fragment, or a derivative thereof) of the present invention can be completely synthesized by chemical synthesis. This DNA sequence can then be introduced into various DNA molecules (e.g., vectors) and cells in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the human LYG2 of the present invention has a human-derived natural amino acid sequence, it is expected to have higher activity and / or lower side effects when administered to humans compared to homologous proteins derived from other species ( (Eg, less or not immunogenic in humans).
  • the present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.
  • the experimental methods without specific conditions in the following examples are generally performed according to the conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions.
  • EST Using the obtained EST as a probe, the EST database is searched again to obtain an EST sequence that partially overlaps with it and extends 5 'or 3'. These newly obtained ESTs were aligned with the probe sequences into a contig. Search the EST database with the 5 'and 3' ends of this contig and repeat the above steps until you get an EST contig that can no longer be extended. 2) Primer amplification
  • primers were designed.
  • a human testis ⁇ gtlO cDNA library purchased from Clontech
  • a pair of oligonucleotides were used as primers-A1: 5'- GTAAGGTTGCAAACAAGGTCCTG-3 '(SEQ ID NO. 3) as the forward primer
  • the oligonucleotide Acid B1 5'-TCACACTGGTCTTCAGTGGTCTC-3 '(SEQ ID NO. 4) is a reverse primer
  • PCR is performed.
  • the PCR conditions were 93 V for 4 minutes, followed by 93 V for 1 minute and 70 ° C. 35 cycles were performed at 1 minute at C and 72 minutes at 72 V, and the last 72 minutes was extended for 5 minutes.
  • the PCR fragment obtained by electrophoresis was a 846bp target fragment.
  • the PCR amplification product obtained above was ligated with the pGEM-T® vector (Promega), transformed into E. coli JM103, the plasmid was extracted with QIAprep Plasmid Kit (QIAGEN), and the inserts were inserted with the double-stranded nested deletion kit (Pharmacia). A targeted series of deletions is performed, and then the deletions are quickly identified and sorted by PCR. Sequenced truncated deletions were sequenced using the SequiTherm EXCELTM DNA Sequencing Kit (Epicentre Technologies). Finally, the sequence was spliced using computer software to obtain a full-length cDNA sequence, a total of 846 bp. The detailed sequence is shown in SEQ ID NO.
  • Computer technology using BLAST is used to search databases for, for example, Genbank, the same or related molecules. This analysis is much faster than a method based on multiple membrane hybridizations. In addition, the sensitivity of the computer search can be adjusted.
  • the search results revealed that the human LYG2 of the present invention shows high homology with members of the G-type lysozyme family.
  • Northern analysis is a laboratory technique used to detect gene transcription. It involves: hybridizing a labeled nucleotide sequence to a membrane containing RNA extracted from a particular cell or tissue.
  • the hybridization probe designed according to SEQ ID NO: 2 can be used to search its cDNA sequence, genomic sequence and mRNA sequence. Although this method is designed to label these 23 or so oligonucleotides, it can also be applied to longer nucleotide sequences in essence. Oligonucleotide probes are designed using software such as PCGENE. For labeling, a system of 50 pmol of free nucleotides, 250 mCi of Y- 32P- labeled adenosine triphosphate and T4 polynucleotide kinase was used. The labeled oligonucleotides were purified on a G-25 dextran column (Amersham Pharmacia Biotech). Fix the purified probe to the hybridization membrane, wash the membrane, and reduce the background to 7-10 cpm.
  • Microarrays also known as DNA chips.
  • the chip can be obtained by immobilizing a sample on a substrate using a series of chemical methods and inkjet technology. Common methods include radiation, chemistry, thermodynamics, mechanical methods, etc. Shapes, bars, etc.
  • a typical chip usually contains a certain number of components, which can be prepared by hand or with appropriate equipment. After the hybridization reaction, the unbound probe is washed away, and then a scanner is used to detect the components and the degree of the reaction that occurred. The complementarity and binding amount of the probe to each chip element can be judged by analyzing the scanned image.
  • Full-length cDNA, EST, or gene fragments can be used as substrate-fixed samples.
  • the fragments suitable for hybridization can be selected by using some well-known biological software such as LASERGENE SOFTWARE (DNASTAR). These full-length cDNAs, ESTs, fragments related to the nucleic acid sequence of the present invention, or cDNA libraries related to this invention
  • the randomly selected fragments are arranged in an orderly manner on a carrier such as glass.
  • the method for fixing cDNA to a glass slide is: UV cross-linking, thermal, chemical treatment, and drying (Schena, M. et al. (1995) Science 270: 467-470: and Shalon, D. et al. (1996) Genome Res. 6: 639-645.) Etc. After the probe is fluorescently labeled, it is then hybridized with the anchoring reaction element. The results of the hybridization are analyzed by the aforementioned method.
  • Example 6 Complementary polynucleotide fragments
  • the complementary sequence or complementary fragment of LYG2 can be used to detect, reduce or inhibit the expression of natural LYG2.
  • the oligonucleotides mentioned in Example 4 are 15-30 base pairs. In principle, this method can also be used for smaller or larger nucleotide fragments.
  • LYG2 complementary oligonucleotides and coding sequences can be designed with software 0LIG0 4. 06. Designing a specific complementary fragment at the 5 'end of LYG2 can prevent the promoter from binding to LYG2 to inhibit transcription, and designing a complementary polynucleotide fragment can also prevent it from binding to the ribosomal subunit and LYG2 coding transcript to prevent protein translation.
  • LYG2 The expression and purification of LYG2 is based on the use of bacterial or viral expression systems.
  • the cDNA of LYG2 is first ligated to a vector containing an antibiotic-resistant and inducible high-efficiency promoter.
  • a promoter includes at least a trp-lac (tac) fusion promoter and a lac operon regulatory unit T5 or T7 phage promoter and so on.
  • tac trp-lac
  • T5 or T7 phage promoter a lac operon regulatory unit
  • the baculovirus polyhedrin gene is replaced by the LYG2 cDNA sequence through homologous recombination or bacterial-mediated transposition.
  • the replaced virus remains infectious and the strong promoter of the polykeratin gene causes transcription of the LYG2 gene Can be performed efficiently.
  • the bacterial or viral infectivity and the powerful function of the recombinant promoter ensure high expression of LYG2.
  • Recombinant baculoviruses are mostly used to infect insect Sf9 cells. If the genetic characteristics of the baculoviruses are modified, they can also be used to infect human liver (parenchymal) cells (Engelhard, EK et al. (1994) Proc Natl. Acad. Sci. USA 91: 3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945.).
  • LYG2 is usually combined with GST or peptide tags (such as FLAG, 6-His, etc.) to form a fusion protein, so that the recombinant fusion protein can be quickly and easily extracted and purified from the bacterial lysate.
  • GST is an enzyme with a molecular weight of approximately 26 kDa extracted from Schistosoma japonicum. It can be used to extract fusion proteins in the presence of solidified glutathione to ensure that its activity and antigenicity are not destroyed (Amersham Pharmacia Biotech). After the fusion protein is extracted, GST and LYG2 can be separated at specific sites according to the conventional operation manual.
  • FLAG is a polypeptide consisting of 8 amino acid residues, which can be obtained by using commercially available monoclonal or polyclonal antibodies against FLAG Specific immunoaffinity between the antibody and FLAG to purify the fusion protein (Eastman Kodak).
  • 6-His is a polypeptide containing 6 consecutive histidines, and the fusion protein can be extracted by utilizing its characteristic of specifically attaching to metal chelating resin.
  • the LYG2 purified by the above method can be directly used in activity detection experiments.
  • Example 8 Activity verification
  • the protein activity of LYG2 can be verified by measuring its ability to lyse micrococcal cells (Enzymatic Assay of Lysozyme 1, Sigma Aldrich, St. Louis Mo.). At 25 ° C for an appropriate amount of the lyophilized powder was suspended in Micrococcus 66mM, P H6. 24 potassium phosphate buffer (buffer A), the concentration was maintained at about 0.015%. 5ml ⁇ ⁇ suspending the above suspension into a cuvette, maintaining a constant temperature of 25 ° C, with a constant temperature spectrophotometer to determine its absorbance at 450nra, until it is constant between 0. 6-0. 7 with 2. 5ml of Buffer A was used as a blank control.
  • LYG2 solution prepared with cold buffer A
  • buffer A was added to the cuvette as a control, immediately reversed and mixed, and continuous measurement Absorbance at 450 nm in 5 minutes.
  • the solution became clear when the bacteria were lysed, so their absorbance at 450nm also decreased.
  • the rate of decrease of the absorbance of the solution in the sample tube at 450nra is proportional to the concentration of the original LYG2 solution.
  • the cDNA is first ligated to a mammalian vector containing a strong promoter, such as PCMV SPORT (Life technology), PCR3.1 (Invitrogen, Carlsbad, Calif.) And the like containing a cytomegalovirus promoter. 5-10ng recombinant vector was transfected into a human cell line by liposome or electroporation. The best choice was endothelial cells or hematopoietic stem cells. At the same time, 1-2ng of the plasmid containing the coding protein coding sequence was co-transfected. Labeled proteins can be used to distinguish transfected cells from untransfected cells. Such proteins include GFP, CD64, and GFP-CD64 fusion proteins.
  • Flow cytometry is a technology based on laser that can automatically detect cell traits. It can also know whether cDNA on a recombinant vector is expressed. It can detect transfected cells expressing GFP or CD64-GFP, as well as detect cell traits, such as apoptotic status. It can also detect changes in cells that occur before or with cell death by detecting the number of fluorescent molecules and the intensity of the fluorescence.
  • the effect of LYG2 expression on cells can also be obtained by detecting cells containing GFP or GFP-CD64 fusion protein coding sequences.
  • CD64 or GFP-CD64 is expressed on the cell surface and usually binds to conserved regions of human IgG or CD64 antibodies.
  • magnetic beads that bind human IgG or CD64 can effectively distinguish transfected and untransfected cells (DYNAL, Lake Success, NY).
  • isolation can be performed using conventional techniques in the art.
  • the expression of mRNA encoding LYG2 and other related genes can be detected by Northern hybridization or gene chip technology.
  • Example 10 Preparation of antibodies LYG2 purified by PAGE gel electrophoresis can be used to immunize rabbits to produce antibodies.
  • the highly immunogenic sites of the amino acid sequence of LYG2 obtained by the analysis of the LASERGENE software can be used to synthesize corresponding oligopeptides and obtain antibodies by conventional methods in the art.
  • Appropriate epitopes can be selected by conventional methods, such as the C-terminus or the hydrophilic region.
  • peptides of 15 amino acids in length can be synthesized with an ABI 43 / A peptide synthesizer and can be combined with KLH to increase immunogenicity (by binding to MBS).
  • the complex of peptide-KLH binding was emulsified with complete Freund's adjuvant to immunize rabbits.
  • the obtained antiserum can be detected by immune serum reaction.
  • the above polypeptide is connected with a plastic carrier blocked with 1% BSA, and then mixed with rabbit antiserum for reaction, washed, and then with radioiodine-labeled antirabbit serum Goat antibody reacts.
  • Example 11 Purification of natural LYG2 with specific antibodies
  • Both natural and recombinant LYG2 can be purified by immunoaffinity chromatography.
  • Columns can be constructed by covalently combining antibodies against LYG2 with active chromatography resins, such as SEPHAR0SE resins (Amersham Pharmacia Biotech) activated by CNBr. After covalent bonding, the resin can be blocked and washed according to the instruction manual.
  • LYG2 is bound to the column.
  • the binding strength of LYG2 to the column must be guaranteed to the greatest extent.
  • a high ionic strength cleaning agent is used as the eluent.
  • the LYG2 bound to the column needs to be eluted.
  • a reagent that can dissociate the binding force between LYG2 and its antibody such as a PH2. 0-3. 0 chaotropic agent, is highly concentrated. Urea or thiocyanate ion.

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Abstract

La présente invention concerne un nouveau lysozime de type G humain (LYG2) et le polynucléotide codant le LYG2. L'invention concerne également les vecteurs d'expression, des cellules hôtes, les anticorps, les agonistes et les antagonistes correspondants. L'invention concerne aussi la méthode de diagnostic, de traitement et de prévention des maladies liées à l'expression du LYG2.
PCT/CN2001/001176 2001-03-02 2001-07-17 Lysozime de type g humain, sa sequence codante, son procede de preparation et ses utilisations WO2002070715A1 (fr)

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CN100344762C (zh) * 2004-10-22 2007-10-24 中国科学院海洋研究所 栉孔扇贝g型溶菌酶基因和编码蛋白及其克隆方法
CN103073632B (zh) * 2011-10-26 2016-08-03 北京大学 具有抗感染和抗肿瘤活性的潜在新细胞因子lyg1及其应用
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